Project summary: The initial interaction of HIV-1 with the host cells is mediated by the virus envelope (Env) spike, each of which is composed of three gp120-gp41 heterodimers. Since the virus Env is the only viral antigen present on the virus surface, it is the sole target for the host antibody (Ab) responses. One hallmark of the HIV-1 Env is its heavy glycosylation. Indeed, glycans comprise half of the molecular mass of the gp120 subunit. The N-linked glycans are essential for the proper folding of the Env, and the >25 N-glycans shrouding each of the gp120 subunit are known to shield Ab epitopes. However, very little is known about the mechanisms and factors that influence the glycan occupancy and the types of glycans (high-mannose, hybrid, complex) decorating the virus Env. Moreover, the importance of N-linked glycans and especially their sugar compositions in regulating the infectivity and transmissibility of the virus is not fully understood. In this proposal we focus on studying the contribution of the Env signal sequence (SS) variations in modulating HIV Env functions and glycosylation. The SS has been shown to play a crucial role in the biosynthesis, glycosylation and conformational folding of glycoproteins in general. Notably, the HIV Env SS is as highly variable as the Env variable loops, and prominent sequence differences are observed between SS of T/F vs. acute vs. chronic viruses, and between SS of Ab-sensitive Tier 1 viruses and Ab-resistant Tier 2 and Tier 3 viruses. Our preliminary data show that a single amino-acid change in the Env-SS is sufficient to drastically alter HIV-1 Env recognition and virus neutralization by MAbs that target often masked epitopes on the V2 and V3 loops. The Env-SS mutations also affect virus trans-infection via DC-SIGN, which binds to N- glycans on the HIV-1 Env. Hence, we propose an overall hypothesis that the Env SS is an active modulator of HIV-1 Env functions. Mutations in the Env SS affect virus infectivity, transmission via DC- SIGN and across epithelial barrier, and antibody recognition and neutralization by modulating the N- glycan occupancy and sugar compositions of the virus Env. To test this hypothesis, we will determine the impact of HIV Env SS polymorphisms found in a T/F Tier 2 virus vs. other viruses (T/F, chronic, Tier 1, Tier 2) in modulating the virus infectivity, transmission, antigenicity, and neutralization using cell-based and immunochemical assays (Aim 1). We will further evaluate whether Env SS variations are associated with alterations of N-glycan compositions using oligosaccharide-specific lectins and high energy C-trap dissociation mass spectrometry (HCD-MS) (Aim 2). These studies will lead us to a better understanding about the contribution of SS polymorphisms in influencing HIV-host interactions and provide valuable data for designing novel strategies to develop more effective Env immunogens for HIV vaccines.